Grantee Research Project Results
Final Report: The Biochemistry and Molecular Biology of Microbial Selenate and Arsenate Transformation
EPA Grant Number: R826105Title: The Biochemistry and Molecular Biology of Microbial Selenate and Arsenate Transformation
Investigators: Stolz, John F.
Institution: Duquesne University
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1997 through September 30, 2000
Project Amount: $273,119
RFA: Exploratory Research - Environmental Biology (1997) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Aquatic Ecosystems
Objective:
The mobilization of toxic elements and heavy metals in the environment can be strongly influenced by microbial activity. Sulfurospirillum barnesii has the ability to couple the oxidation of organic matter to the reduction of selenate and arsenate, compounds that have become significant environmental toxins. The hypothesis to be tested is that S. barnesii has separate pathways for arsenate and selenate reduction, but the selenate reductase is a less substrate-specific enzyme, capable of reducing a wide range of substrates. The objective of this project is to purify and characterize (Km, Vmax, substrate specificity, absorption spectrum, mid-point potential) the two reductases and associated cytochromes, and develop biochemical and molecular probes based on the selenate reductase that can be used to detect S. barnesii in nature. The probes also will be used to determine the relatedness of selenate reductases from other bacterial species.Summary/Accomplishments (Outputs/Outcomes):
This work is part of a continuing collaboration between the PI and Ronald S. Oremland of the U.S. Geological Survey in Menlo Park, CA. When this grant was initially funded, there were only four species of arsenate-respiring bacteria and two species of selenate-respiring bacteria known. Included among them was the arsenate and selenate-respiring bacterium Geospirillum barnesii strain SES-3. As part of this project, we were able to complete the characterization of this strain and a closely related strain that respires arsenate but not selenate. They were officially described as members of the Sulfurospirillum clade in the epsilon Proteobacteria and given the names Sulfurospirillum barnesii and S. arsenophilum (Stolz, et al., 1999; 2002). Since then, Dr. Oremland's group has isolated four species of bacteria capable of respiring oxyanions of arsenic and/or selenium. My laboratory has contributed to these efforts through 16S rRNA sequencing and phylogenetic analysis, ultrastructural characterization (i.e., transmission electron microscopy), and biochemical analysis. Two strains of haloalkaliphilic bacilli were isolated from Mono Lake, CA. Bacillus arsenicoselenatis strain E1H is a spore forming, strict anaerobic low G+C bacillus that is able to respire both arsenate and selenate. Bacillus selenitireducens strain MLS10 is a non-spore-forming low G+C bacillus that also is able to respire arsenate and is the first organism described to respire selenite (Switzer Blum, et al., 1998). Selenihalobacter shriftii strain DSSe-1 is a selenate-respiring halophilic gram negative bacterium belonging to the Haloanaerobacteria that was isolated from Dead Sea sediments (Switzer Blum, et al., 2001). A species of Citrobacter (as determined by 16S rRNA analysis) was isolated from termite hind-gut that is capable of both selenate and arsenate respiration (Switzer Blum, et al., unpublished). In my own laboratory, we have isolated and characterized two freshwater bacteria from Ohio River sediments. Strain Oh2F is a strictly anaerobic spore-forming low G+C gram positive that is most closely related to the dehalogenating bacteria of the genus Desulfitobacterium (Kuchan, 2000, Master's thesis). It is able to grow using formate as the electron donor and carbon source, and selenate (20 mM) as the electron acceptor. Strain OhILAs also is a strictly anaerobic spore forming low G+C gram positive, but it is most closely related to species of the genus Clostridium. It is capable of growing on acetate and arsenate. Astonishingly, it grows very well on 30 mM arsenate and may even be able to respire arsenite (Dawson, Master's thesis in preparation). Today, the number of species of arsenate-respiring bacteria has almost tripled and includes two members of the Crenarchaeota, Pyrobaculum arsenaticum and P. aerophilum. The number of selenate-respiring bacteria has more than tripled. These results have contributed to a better understanding of the diversity of species capable of respiring arsenic and selenium oxyanions as well as the wide range of environments in which they inhabit.Given the limited number of species at the time, it made absolute sense to begin this study of the biochemistry of arsenate and selenate respiration with S. barnesii. Although our original hypothesis that the selenate and arsenate reductases were separate enzymes was confirmed, our assumption that they were molybdo-enzymes similar to DMSO reductase and nitrate reductase, however, turned out to be wrong. In fact, the arsenate reductase complex contains two proteins that are new to science. This has resulted in the biochemical results not being published in a timely manner and delayed the completion of the work on the selenate reductase. It has, however, facilitated the completion a major goal of this work, the development of species-specific biochemical and molecular probes for the detection of S. barnesii in natural environments.
The preliminary work on the arsenate reductase complex suggested that it was comprised of three subunits with polypeptides of 65, 30, and 20 kDa. N-terminal amino acid sequence analysis revealed that the 65 kDa polypeptide was the large subunit of a nickel-iron hydrogenase. This was later confirmed by metals analysis and Western blot analysis with antibodies specific to the large subunit of the Ni-Fe hydrogenase from Wolinella succinogenes (courtesy of A. Kroeger). Neither the 30 or 20 kDa polypeptides, however, matched any known protein or gene sequences in the databases. Subsequently, we employed several different types of column chromatography including size exclusion, hydrophobic interaction, hydroxyapatite, and ion exchange to purify the complex. Regardless of how it was purified, the complex contains the 30 and 20 kDa polypeptides. It exhibits a Km of 47 M for arsenate with a Vmax for arsenate reduction of approximately 0.23 mol min-1 mg-1 at 25 ?C and pH 7.2. NADH, FMN, and methyl viologen can be used as electron donors. Using the N-terminal amino acid sequence data, degenerate PCR primers were designed. (The gene sequencing was done by Dianne Newman, who began the project when she visited my laboratory in the summer of 1997 and has continued the work at the California Institute of Technology where she has a faculty position). A portion of the ars operon was amplified by PCR using a forward primer based on the 30 kDa protein sequence and a reverse primer based on the 20 kDa polypeptide (Newman, unpublished). Thus, the two proteins are indeed linked, as the genes encoding them are on the same operon. The gene encoding the 30 kDa polypeptide (for which all but the upstream promoter region was obtained) is devoid of conventional metal binding motifs but has four histidine residues as possible arsenic binding sites. The 20 kDa polypeptide forms a dimer in the native state (based on protein sequence data) and has a leader sequence indicative of an exported protein. The complete open reading frame has not, however, been completed as yet. In addition, two small open reading frames lie in between genes encoding the 30 and 22 kDa proteins. BLAST searches of published databases (i.e., GenBank, EMBL) and 25 of the near complete bacterial genomic projects have failed to find homologues to the 30 kDa gene or polypeptide. Thus, it was an excellent candidate for a species-specific probe for S. barnesii. We have successfully raised polyclonal antibodies against this protein. Western blot analysis is currently underway to determine the specificity to other arsenate-respiring bacteria and the levels of expression of the 30 kDa polypeptide in cells of S. barnesii grown on different substrates (i.e., selenate, nitrate, thiosulfate, fumarate, etc.). We also are using the whole gene as a molecular probe for fluorescence in situ hybridization (FISH) and Southern blot analysis. In addition, two new sets of PCR primers have been designed to amplify the gene product from natural samples. The final revisions are being made to the PNAS paper, and it should be resubmitted this fall. A paper describing the development and application of the biochemical and molecular probes also is planned.
Work continues on the purification and characterization of the selenate reductase. Again, this enzyme does not appear to be a typical molybdo-enzyme (i.e., nitrate reductase). A complex has recently been purified using ion exchange and size exclusion chromatography. It is comprised of two major polypeptides of 93 and 45 kDa. The enzyme complex has an apparent Km for selenate of 13.1 mM and a Vmax of 28.2 mol of methyl viologen oxidized min-1 mg-1. We anticipate the completion of this work in the later part of 2001 with publication in the spring of 2002. An interesting aspect of selenate metabolism in S. barnesii is that it can simultaneously reduce micromolar concentrations of selenate in the presence of millimolar concentrations of nitrate (Oremland, et al., 1999). This is very important when considering bioremediation strategies for agricultural soils that have a similar ratio of selenate to nitrate concentration. It appears that S. barnesii has constitutive selenate and nitrate reductase activity as well as inducible high affinity nitrate and selenate reductases. More significantly, Oremland's group, using a 75Se-tracer, have shown that this organism can lower the ambient Se(VI) concentrations (by reduction to Se(0)) to levels in compliance with the current maximum contaminant level allowable for selenium (Oremland, et al., 1999).
An important aspect of this grant was the opportunity to spend several weeks in the laboratory of my collaborator, Ron Oremland, during my sabbatical in the fall of 1998. Several projects were initiated at that time, and it is clear from the publication list that this collaboration has been and continues to be quite productive. I am pleased to report that this work is being continued under grants from the USDA (Proposal # 0569) and most recently from the USGS-NIWR grant program (Proposal 2001PA721G). The probes that have been developed in this study are being applied in studies of arsenate-contaminated sites in Pennsylvania (USGS-NIWR grant) and in future studies of microbial arsenate-reduction in animal GI tracts. These tools will be most helpful in determining the role of microorganisms in the mobilization and speciation of arsenic and selenium in the environment.
Journal Articles on this Report : 5 Displayed | Download in RIS Format
Other project views: | All 12 publications | 8 publications in selected types | All 5 journal articles |
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Blum JS, Bindi AB, Buzzelli J, Stolz JF, Oremland RS. Bacillus arsenicoselenatis, sp nov, and Bacillus selenitireducens, sp nov: two haloalkaliphiles from Mono Lake, California that respire oxyanions of selenium and arsenic. Archives of Microbiology 1998;171(1):19-30. |
R826105 (1999) R826105 (Final) |
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Blum JS, Stolz JF, Oren A, Oremland RS. Selenihalanaerobacter shriftii gen. nov., sp nov., a halophilic anaerobe from Dead Sea sediments that respires selenate. Archives of Microbiology 2001;175(3):208-219. |
R826105 (2000) R826105 (Final) |
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Oremland RS, Blum JS, Bindi AB, Dowdle PR, Herbel M, Stolz JF. Simultaneous reduction of nitrate and selenate by cell suspensions of Selenium-respiring bacteria. Applied Environmental Microbiology 1999;65(10):4385-4392. |
R826105 (1999) R826105 (2000) R826105 (Final) |
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Stolz JF, Oremland RS. Bacterial respiration of arsenic and selenium. FEMS Microbiology Reviews, Volume 23, Issue 5, October 1999, Pages 615-627. |
R826105 (1999) R826105 (2000) R826105 (Final) |
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Stolz JF, Ellis DJ, Blum JS, Ahmann D, Lovley DR, Oremland RS. Sulfurospirillum barnesii sp nov and Sulfurospirillum arsenophilum sp nov., new members of the Sulfurospirillum clade of the epsilon Proteobacteria. International Journal of Systematic Bacteriology 1999;49(Part 3):1177-1180. |
R826105 (1999) R826105 (Final) |
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Supplemental Keywords:
heavy metals, ecology, soil, water, enzymes, indicators, bioremediation., Scientific Discipline, Waste, Ecosystem Protection/Environmental Exposure & Risk, Environmental Microbiology, Fate & Transport, Biochemistry, Bioremediation, Ecological Risk Assessment, Molecular Biology/Genetics, microbiology, microbial selenate, fate and transport, aerobic degradation, biodegradation, arsenate compounds, oligonucleotide probes, biotechnology, reductases, sulfurospirillum barnesii, oligobacteria, heavy metalsRelevant Websites:
http://www.home.cc.duq.edu/~stolz/arsenic.htmlProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.